Doctor of Philosophy, California Institute of Technology (2011)
Doctor of Medicine, University of California Los Angeles (2013)
Bachelor of Science, Massachusetts Institute of Technology (2003)
Markus Covert, Postdoctoral Faculty Sponsor
A Single-Molecule Hershey-Chase Experiment
2012; 22 (14): 1339-1343
Ever since Hershey and Chase used phages to establish DNA as the carrier of genetic information in 1952, the precise mechanisms of phage DNA translocation have been a mystery. Although bulk measurements have set a timescale for in vivo DNA translocation during bacteriophage infection, measurements of DNA ejection by single bacteriophages have only been made in vitro. Here, we present direct visualization of single bacteriophages infecting individual Escherichia coli cells. For bacteriophage λ, we establish a mean ejection time of roughly 5 min with significant cell-to-cell variability, including pausing events. In contrast, corresponding in vitro single-molecule ejections are more uniform and finish within 10 s. Our data reveal that when plotted against the amount of DNA ejected, the velocity of ejection for two different genome lengths collapses onto a single curve. This suggests that in vivo ejections are controlled by the amount of DNA ejected. In contrast, in vitro DNA ejections are governed by the amount of DNA left inside the capsid. This analysis provides evidence against a purely intrastrand repulsion-based mechanism and suggests that cell-internal processes dominate. This provides a picture of the early stages of phage infection and sheds light on the problem of polymer translocation.
View details for DOI 10.1016/j.cub.2012.05.023
View details for Web of Science ID 000306823700025
View details for PubMedID 22727695
Ion-Dependent Dynamics of DNA Ejections for Bacteriophage lambda
2010; 99 (4): 1101-1109
We studied the control parameters that govern the dynamics of in vitro DNA ejection in bacteriophage lambda. Previous work demonstrated that bacteriophage DNA is highly pressurized, and this pressure has been hypothesized to help drive DNA ejection. Ions influence this process by screening charges on DNA; however, a systematic variation of salt concentrations to explore these effects has not been undertaken. To study the nature of the forces driving DNA ejection, we performed in vitro measurements of DNA ejection in bulk and at the single-phage level. We present measurements on the dynamics of ejection and on the self-repulsion force driving ejection. We examine the role of ion concentration and identity in both measurements, and show that the charge of counterions is an important control parameter. These measurements show that the mobility of ejecting DNA is independent of ionic concentrations for a given amount of DNA in the capsid. We also present evidence that phage DNA forms loops during ejection, and confirm that this effect occurs using optical tweezers.
View details for DOI 10.1016/j.bpj.2010.06.024
View details for Web of Science ID 000281103200013
View details for PubMedID 20712993
Biochemistry on a Leash: The Roles of Tether Length and Geometry in Signal Integration Proteins
2009; 96 (4): 1275-1292
We use statistical mechanics and simple ideas from polymer physics to develop a quantitative model of proteins whose activity is controlled by flexibly tethered ligands and receptors. We predict how the properties of tethers influence the function of these proteins and demonstrate how their tether length dependence can be exploited to construct proteins whose integration of multiple signals can be tuned. One case study to which we apply these ideas is that of the Wiskott-Aldrich Syndrome Proteins as activators of actin polymerization. More generally, tethered ligands competing with those free in solution are common phenomena in biology, making this an important specific example of a widespread biological idea.
View details for DOI 10.1016/j.bpj.2008.10.052
View details for Web of Science ID 000266377800006
View details for PubMedID 19217847